PROJECT SUMMARY The DNA replication machinery is constantly challenged by impediments that stall the replication fork. Replication stress and stalled forks are a major source of genomic instability, which underlies a number of diseases including cancer. Replication-repair pathways known as the replication stress response serve to stabilize and restart damaged forks. However, the molecular mechanisms of these pathways are poorly understood, in part because of a dearth of structural information for the proteins involved. Our long term goal is to understand the molecular mechanisms of the replication stress response and how the pathways are interconnected to ensure faithful completion of DNA replication. Our strategy is to couple structural information of the enzymes and multi-protein complexes operating at the replication-repair interface with their biochemistry and cellular functions. We are currently focused on three poorly understood activities at stalled forks?(1) fork reversal and template switching as a mechanism to stabilize damaged forks and restart replication, (2) protection of labile abasic (AP) sites from strand cleavage or mutagenic bypass, and (3) priming of DNA synthesis. Fork reversal by the ATP-dependent DNA translocases HLTF, SMARCAL1, and ZRANB3 involves remodeling of stalled fork into four-way junctions to prevent fork collapse and facilitate replication restart. Our work will address critical gaps in knowledge related to how these enzymes provide unique repair activities at damaged forks, their mechanisms of fork reversal, and how the ubiquitin ligase and DNA remodeling activities of HLTF are coordinated and regulate fork reversal in cells. Secondly, we are working to understand how the SOS Response Associated Peptidase (SRAP) protein HMCES forms a stable DNA-protein crosslink (DPC) with AP sites in ssDNA as a means to protect them from error-prone polymerases and nucleases during replication. AP sites are the most abundant form of DNA damage and thus it is critical that we understand how cells deal with these potent replication blocks. Our recent structure of a SRAP DPC forms the basis for further experiments to understand the chemical biology behind this novel repair pathway. Third, DNA polymerase ?-primase (pol-prim) is a core component of the eukaryotic replisome that initiates de novo DNA synthesis at every Okazaki fragment by synthesizing RNA-DNA primers of defined length. Despite the importance of this critical activity at the replication fork, its mechanism of action is unknown. We are addressing this gap in knowledge by trapping complexes of pol-prim with relevant nucleic acid substrates and intermediates at various stages of its catalytic cycle and visualizing conformational states by electron microscopy and biophysical approaches. Fundamental knowledge of the conformational dynamics that occur in pol-prim during de novo DNA synthesis will be the first step toward understanding the coordination of enzymatic activities at stalled forks.